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M. Doat, C. Andrieu, M. Halhal, F. Behar–Cohen; Transpalpebral Iontophoresis Combined With Oligonucleotides Intravitreous Injection Allows the Targeting of Photoreceptor Cells in vivo . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5196.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To optimize photoreceptors oligonucleotides (ODNs) delivery using saline transpalpebral iontophoresis. Methods:Saline transpalpebral iontophoresis associated with intravitreous injection of ODNs encoding for the sense wild type ß–PDE gene sequence were applied to rd1/rd1 mice homozygous for the nonsense mutation (amino acid position 347) in the ß–PDE gene at post natal day 7 (PN7). Various treatment parameters were tested. Cationic or anionic transpalpebral iontophoresis (respectively positive or negative electrode connected to the eyelids) was performed immediately before or after the intravitreous injection of ODN using a current of 1.5 mA for 5 min (4 eyes for each combination). Conditions with increased intensity and duration of treatment (2.5 mA for 10 min) or lower current intensities (0.5 or 1 mA for 5 min) were also tested (4 eyes for each condition). The effect of timing (1 or 3 hours) between iontophoresis and ODN intravitreous injection was carried out (4 eyes for each condition). For the control groups, rd1/rd1 mice at PN7 received either iontophoresis followed by PBS injection, ODN injection without iontophoresis or, had no treatment (4 eyes for each condition). Fluorescence intensity of the outer nuclear layer fluorescence was quantified by image analysis of cryosections. Microphotographs taken with a fixed exposure time and expressed in mean pixel brightness ± SD served as the basis for this analysis. Results:All conditions of transpalpebral saline iontophoresis increase the penetration of ODNs in the retinal layers when compared to injection without iontophoresis. Intact ODNs were extracted from the retina at one hour after treatment. Photoreceptors targeting is significantly increased when the saline transpalpebral iontophoresis is applied before ODN injection as compared with its application after ODN injection. Anionic iontophoresis is more efficient than cationic iontophoresis. Of all tested conditions, optimal photoreceptor targeting is achieved with application of transpalpebral saline anionic iontophoresis using 1.5 mA for 5 min before ODN injection. The facilitation of intraocular penetration by iontophoresis remains apparent at least for 3 hours. No intraocular tissue damage is observed after the iontophoresis application. Conclusions: : Saline transpalpebral iontophoresis performed under specific conditions is a safe method for increased delivery efficiency of ODNs to photoreceptors. This technique can be applied to enhance targeted gene repair in photoreceptor cells.
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